Luminescence is generally the emission of light that does not derive energy from the temperature of the emitting body. Luminescence may be caused by chemical, biochemical, or crystallographic changes, the motions of subatomic particles, or radiation-induced excitation of an atomic or molecular system. Luminescence quenching refers to any process which can decrease the luminescence intensity of a given luminophore. A variety of processes can result in luminescence quenching, such as excited state reactions, energy transfer, complex formation and collisional quenching.
The luminescence quenching process, especially the fluorescence quenching process, through energy transfer has been well studied. When a first fluorophore is excited and transfers its absorbed energy to a second fluorophore, the energy transfer results in fluorescent signal at the emission wavelength of the second fluorophore. However, where the second fluorophore shows no fluorescence, the absorbed energy does not result in fluorescence emission, and the first fluorophore is said to be “quenched”. Similarly, energy transfer can also be utilized to quench the emission of other luminescent donors such as phosphorescent and chemiluminescent donors.
The use of a variety of dyes containing at least a luminophore to quench luminescence such as fluorescence is known in the art. The application of luminescence quenching to analyze biological systems is also well-studied. However, there is always a need for luminescence quenchers having different absorption properties to meet the various requirements of new advances in this field.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generally known to scientists as very small inorganic or organic molecules with high reactivity. There are various forms of ROS and RNS including free radicals such as superoxide radical, hydroxyl radical, nitric oxide, nitrogen dioxide and organic peroxyl radical as well as non-radical species such as hydrogen peroxide, singlet oxygen, ozone, nitrous acid, peroxynitrite and hypochlorite. ROS and RNS are the by-products of cellular respiration. Under normal conditions ROS and RNS are present in very low levels and play important roles in cell signaling, while during oxidative stresses, ROS and RNS levels increase dramatically, which can cause serious damages to various biological molecules such as protein, lipids and DNA. The excessive generation of ROS and RNS has been implicated in a lot of human diseases, such as cardiovascular diseases, inflammatory diseases, metabolic diseases, cancer and central nervous system diseases. Therefore, there is a strong need for chemicals that can sensitively and selectively measure, detect or screen certain ROS and RNS to address their physiological roles both in vitro and in vivo.
Peroxynitrite and hypochlorite have the strongest oxidizing power among the various forms of ROS and RNS, and their selective detections are highly desirable to clearly explain their critical roles in living organisms. Peroxynitrite (ONOO−) is a short-lived oxidant species that is formed in vivo by the diffusion-controlled reaction (k=0.4−1.9×1010 M−1s−1) of nitric oxide (NO) and superoxide (O2•−) in one to one stoichiometry. The oxidant reactivity of peroxynitrite is highly pH-dependent and both peroxynitrite anion and its protonated form peroxynitrous acid can participate directly in one- and two-electron oxidation reactions with biomolecules. The pathological activity of ONOO− is also related to its reaction with the biologically ubiquitous CO2, thereby producing the highly reactive radicals CO3−• and NO2• in about 35% yield. As a result of this, peroxynitrite can nitrate tyrosine and oxidize proteins, lipids and iron and sulfur clusters of biological molecules. Like other oxidizing agents in living organisms, peroxynitrite and its protonated form have been associated with both beneficial and harmful effects. However, several studies have implicated that peroxynitrite contributes to tissue injury in a number of human diseases such as ischemic reperfusion injury, rheumatoid arthritis, septic shock, multiple sclerosis, atherosclerosis, stroke, inflammatory bowl disease, cancer, and several neurodegenerative diseases (MacMillan-Crow, L. A. et al., Proc. Natl. Acad. Sci. USA 1996, 93, 11853-11858; Rodenas, J. et al., Free Radical. Biol. & Med. 2000, 28, 374; Cuzzocrea, S. et al., Pharmacol Rev. 2001, 53, 135-159; Szabo, C. Toxicol. Lett. 2003, 140, 105-112; White, C. R. et al., Proc. Natl. Acad. Sci. USA 1994, 91, 1044-1048; Lipton, S. A. et al., Nature 1993, 364, 626-632; Pappolla, M. A. et al., J. Neural Transm. 2000, 107, 203-231; Beal, M. F., Free Radical Biol. & Med. 2002, 32, 797-803).
On the other hand, hypochlorite is produced in vivo from hydrogen peroxide and chlorine ions in a chemical reaction catalyzed by the enzyme myeloperoxidase (MPO), which may be secreted by activated phagocytes in zones of inflammation. As a nucleophilic non-radical oxidant, hypochlorite can be used as a microbicidal agent (Thomas, E. L., Infect. Immun., 1979, 23, 522-53 1). Furthermore, neither bacteria nor normal healthy cells can neutralize its toxic effect because they lack the enzymes required for its catalytic detoxification (Lapenna, D. and Cuccurullo, F., Gen. Pharmacol., 1996, 27, 1145-1147).
Generally, hypochlorite can react with some proteins that may play important roles in killing bacterial cells and/or human diseases (Thomas, E. L., Infect. Immun., 1979, 23, 522-531; McKenna, S. M. and Davies, K. J. A., Biochem. J., 1988, 254, 685-692; Hazell, L. J. and Stocker, R., Biochem. J., 1993, 290, 165-172; Hazell, L. J., van den Berg, J. J. and Stocker, R., Biochem. J., 1994, 302, 297-304). When contacting with proteins, hypochlorite may cause damages to the proteins. For example, hypochlorite may alter protein structures, and/or cause fragmentation and dimerization of proteins. As a strong oxidant, hypochlorite can also oxidize low-density lipoproteins (LDL) rapidly. Furthermore, the reaction of hypochlorite with DNA can also result in both chemical modifications and structural changes in DNA (Hawkins, C. L. and Davies, M. J., Chem. Res. Toxicol., 2002, 15, 83-92; Prutz, W. A., Arch. Biochem. Biophys. 1996, 332, 110-120; Arch. Biochem. Biophys. 1998, 349, 183-191; Arch. Biochem. Biophys. 1999, 371, 107-114).
Because of the above-mentioned uses and roles of ROS and RNS, there is a need for methods that detect, measure and/or screen ROS such as hypochlorite and/or RNS such as peroxynitrite, including in vivo detection and measurement.